The invention relates to a method of delivering imaging agents; means therefor including components thereof which have particular, but not exclusive, application in the development of therapies for cancer or coronary heart disease.
Macrophages often comprise 20-60% of the tumour cell mass in breast carcinomas and form intimate contacts with malignant cells. This has long been thought to represent part of the host""s defence mechanisms against the tumour; however, their function at such sites in the body remains an enigma at present as macrophages isolated from human or murine tumours exhibit reduced tumouricidal, phagocvtic and antigen-presenting activities compared to those from normal tissues (1).
Monocites are produced in the bloodstream and extravasate (i.e. exit) into surrounding tissues including such diseased tissues as malignant tumours and atherosclerotic plaques, where they differentiate into macrophages and perform immune, secretory, phagocytic and other functions. Monocytes and macrophages are collectively termed mononuclear phagocytes. As tissue macrophages have a lifespan of 60 to 90 days and the number of macrophages in tumours remains constant, it is believed that there is a constant attachment of monocytes to the tumour endothelium and influx of monocytes into the tumour cell mass.
Hypoxia, that is, very low levels of oxygen, exist only in some forms of diseased tissue (e.g. malignant tumours, ischaemic heart tissue, retinal tissue etc.) (2). Hypoxia and/or hypoglycaemia is thought to occur in growing tumours when the increasing metabolic demands of the rapidly expanding tumour cell population outstrip the supply of oxygen/glucose etc., made available to them by simple diffusion across the tumour mass from vessels in surrounding normal tissues.
Recent and surprising data indicate that once monocytes enter a tumour from the bloodstream, they rapidly differentiate into macrophages and preferentially congregate in hypoxic (i.e. poorly vascularised and necrotic) sites deep within a tumour mass remote from blood vessels. Refer to FIG. 1, which represents a bar chart of the Distribution of Macrophages in Relation to Blood Vessels. Moreover, breast tumours, with more hypoxic/necrotic areas, are more heavily infiltrated with macrophages, which preferentially locate to, or around, the necrotic sites (refer to FIG. 2, which represents a bar chart of the Association of Macrophage Index with Necrosis in Breast Carcinomas). Experimental hypoxia has been shown to induce the production of angiogenic factors by macrophages in vitro (3). Taken together these data could underpin our recent finding, that increased numbers of macrophages in breast tumours equate with increased fatalities in breast cancer (4).
We have also shown recently that human macrophages accumulate specifically in cell layers immediately adjacent to the central areas of necrosis in three-dimensional cultures of human cancer cells. Many previous studies have shown that this viable rim of tumour cells around the necrotic core of such spheroids are severely hypoxic relative to the outer layers of tumour cells in these cultures (5). That macrophages congregate in hypoxic diseased tissues other than malignant tissue has been shown for coronary heart disease (6), as well as such cerebrovascular disorders of the central nervous system as strokes and cerebral malaria (7).
The observation that tumour and other forms of ischaemic tissue are regions of poor oxygenation has lead to the development of a number of techniques to assess oxygen tension in these tissues. Invasive surgical procedures include the insertion of polarographic micro-electrodes into tumour tissue to measure directly the levels of oxygen in a given tissue (2). Non invasive techniques have also been adopted which involve the use of radiopharmaceuticals (eg F-18 Fluoromisonidazole) which bind to hypoxic cells (8). The concentration of the agents are then detected and quantified by methods such as whole body positron emission tomography (PET imaging) (9). A major problem with this imaging technique is that radiopharmaceuticals tend to be neurotoxic due to their lipid solubility. Clearly this problem would be overcome if it were possible to bind these products to a delivery means. A further major problem with this imaging technique is the poor level of resolution achieved by radiopharmaceuticals due to a relatively high background detection in tissues that do not have appreciable levels of hypoxia. An improvement of the level of detection in hypoxia sites can be achieved if sufficient time is allowed for the clearance of the radiopharmaceutical from non target tissues. However this can take several hours to achieve and is therefore not a desirable situation.
The current state of the art describes a number of means to enhance the localisation of imaging agents to hypoxic and/or ischaemic sites. In broad terms current techniques involve the encapsulation of imaging pharmaceuticals within microvesicles. Alternatively the imaging agents can be directly modified to enable either the localisation of the agent to the desired tissue or enhance their detection when the agent accumulates in the target tissue.
Typically microspheres encapsulating an imaging agent are liposomes composed of either pure phospholipid or a mixture of phospholipid and phosphoglyceride. They are advantageous due to the ease with which the microspheres can be produced containing the imaging pharmaceuticals. By altering conditions during manufacture microspheres can be produced that have diameters of less than 200 NH which enables them to be intravenously injected and able to pass through the pulmonary capillary bed. Furthermore the biochemical nature of the liposome confers permeability across blood vessel membranes to access the tumour site or region of ischaemia. Liposomes of this type show high echogenicity both in vitro and in vivo which would be a necessary requirement using techniques of magnetic resonance imaging (11), fluoroscopy and computerised tomography (10).
However this technology does suffer a major disadvantage in that the liposomes lack an intrinsic affinity for the targeted tissue and relies on a local intravenous injection of the liposome composition in the vicinity of the diseased tissue.
What patients require is a rapid and accurate diagnosis of their condition so that an effective treatment regime can be established as quickly and accurately as possible. The development of an effective means of targeting imaging means to hypoxic/ischaemic sites would obviously benefit both clinicians and patients in the diagnosis and treatment of diseases such as cancer and coronary heart disease.
An alternative strategy is to chemically modify an agent that has a natural affinity for tumour/ischaemic tissue to enable the detection of the agent at the targeted tissue.
Monosaccharide derivatives have been used as imaging agents (Patent application no. WO.9634872-A). Glucose levels have been shown to be an important indicator in diagnosis of Alzheimer""s disease, Parkinson dementia, epilepsy, diabetes and myocardial ischaemia. The elevated levels of glucose consumption in tumour or ischaemic tissue has been exploited by using iodinated glucose to identify these regions. Although modified monosaccharides have excellent in vivo stability they have a general biodistribution in the body and problems with optimising the signal to noise ratio during treatment can arise.
The labelling of peptides with technetium-99m and there detection via scintigraphic imaging has been used in the diagnosis of tumours (Patent application no. WO.9310747-A). Peptides are typically composed of 4-100 amino acid residues. The technetium-99m labelled peptides have been successful used to diagnose kidney disorders by scintigraphic imaging. However although imaging peptides have excellent in vivo stability they lack an intrinsic targeting property making resolution somewhat problematic.
More recently the use of a radioactive copper complex of dithiosemicarbazone has been employed to image regions of hypoxia and/or mitochondrial dysfunction (EP-726077-A). The composition is advantageous due to improved permeability and retention in target cells but with a short residence in non-targeted cells. The imaging potential of this composition is improved due to the fact that it is only reduced in tissue containing an excess of electrons (eg tissues that contain dysfinctional mitochondria). However although copper containing dithiocarbazone shows retention in hypoxic tissue there are still significant levels of the composition in non-targeted normoxic tissue thus reducing detection resolution.
Finally, the use of monoclonal antibodies to target radioactive and non radioactive imaging agents has been exploited. The expression of specific membrane proteins has lead to the production of monoclonal antibodies to these membrane proteins to enable the targeting of imaging agents to tumour tissue. However this advantage is offset by the poor access of the tagged antibodies to the tumour tissue.
In summary the compositions that have been described either lack an intrinsic means of targeting the imaging agent to a diseased tissue or have poor access to the sites of hypoxia/ischaemia. This leads to a poor signal to noise ratio resulting in reduced imaging resolution.
It is therefore an object of this invention to identify a means to target imaging agents to regions of hypoxia/ischaemia which exploits the fact that mononuclear phagocytes have an affinity for regions of hypoxia/ischaemia.
The invention in its broadest aspect, comprises the use of mononuclear phagocytes to deliver conventional imaging agents to tissues and especially hypoxic/ischaemic sites.
According to a first aspect of the invention there is therefore provided an imaging means comprising an imaging agent attached to an agent that binds to a cell surface element of a mononuclear phagocyte.
The invention comprises the conjugation of imaging agents to the surface of macrophages via macrophage specific cell surface proteins/receptors (eg CD68, CD87, CSF-1). The imaging agent could be attached via a monoclonal antibody, ideally humanised, to one or more of these cell surface proteins or to a ligand specific for a particular macrophage cell surface marker. The macrophages could be modified either in vivo or ex vivo and reintroduced into the patient to allow macrophage migration into the hypoxic/ischaemic tissue. It may be advantageous, but not always necessary, to use an imaging acent that becomes more readily detected due to the conditions of hypoxia or ischaemia.
In a preferred embodiment of the invention said imaging agent is of a conventional nature such as, without limitation, an imaging pharmaceutical such as a radiopharmaceutical or a technetium-99m peptide.
It is therefore a further object to provide a novel imaging means the detection of which is enhanced by hypoxic/ischaemic conditions.
According to a further aspect of the invention there is provided an imaging means comprising a hypoxia enhanced imaging agent and an agent that binds to a cell surface element of a mononuclear phagocyte.
It will therefore be apparent that the hypoxia enhancing imaging agent will be affected by hypoxic conditions and typically affected so as to lead to enhanced detection in such conditions. A typical example of this sort of agent is described in WO 9634872-A. Moreover, said binding agent, which is typically coupled to said imaging agent, attaches the composition to mononuclear phagocytes and so targets the imaging agents, to sites typically infiltrated by mononuclear phagocytes. Thus in the instance where the said mononuclear phagocytes penetrate hypoxic sites said composition is suitably delivered to such sites and the imaging agent shows enhanced detection.
The invention is elegant in so far as the body""s own mechanisms are exploited for the specific delivery of the imaging agent to regions of hypoxia or ischaemia.
Given the above nature of the invention agents suitable for use in manufacturing the composition will be known to those skilled in the art and therefore the following preferred embodiments are not intended to be exhaustive but rather illustrative.
It may be preferable to conjugate said imaging agent to a carrier molecule that promotes the internalisation of the imaging composition into mononuclear phagocytes. Intemalisation signals include, but are not limited to, plasminogen activation inhibitors (PAI-1 PAI-2) or protease nexin (PN), which bind to, and cause the internalisation of CD87 (the receptor for Urokinase Plasminogen Activator) into monocytes and macrophages.
In a preferred embodiment of the invention said binding agent is adapted to bind to any one or more cell surface mononuclear phagocyte molecules such as antigens or receptors.
Further, said bindina agent may comprise an antibody to any one or more of said molecules such as antigens or receptors, or an effective fragment of said antibody. Alternatively still said binding agent may comprise a suitable ligand either synthetically manufactured or naturally occurring. For example, chemicals such as benzodiazepines and PK1195 bind to a specifc receptor on the surface of macrophages (Zavala and Lenfant (1987) Annals N Y Acad Sin 496, 240-249).
A brief list of those cell surface molecules that may be targeted by said binding agent is as follows; CD87; the receptor for human Colony Stimulating Factor (CSF-1); CD11b; CD3; the scavenger receptor; all or part of the receptor for the various forms of human monocyte chemoattractant protein (MCP-1,2, etc); CD14; mannose or mannose-6-phosphate surface receptors.
According to yet a further aspect of the invention there is provided a delivery system for targeting imaging compositions to hypoxic or ischaemic sites comprising an imaging agent and, optionally, an agent for controlling the functional effectiveness thereof, and coupled thereto, a binding agent for a cell surface molecule of a mononuclear phagocyte.
According to yet a further aspect of the invention there is provided a method for targeting imaging agents to hypoxic or ischaemic sites comprising;
(i) coupling at least one of said agents to a binding agent of a cell surface molecule expressed by a mononuclear phagocyte;
(ii) exposing said coupled agents to the mononuclear phagocytes; and
(iii) allowing the said phagocytes to migrate under conditions that support migration in vivo.
According to yet a further aspect to the invention there is provided a method for imaging hypoxic or ischaemic sites comprising administering to an individual to be treated the imaging means of the invention.
According to yet a further aspect of the invention there is provided mononuclear phagocytes having coupled thereto, or internalised therein, an imaging agent and an agent that is adapted to bind to a mononuclear phagocyte ligand which is typically found on the cell surface of the said mononuclear phagocyte.
In essence the invention describes the use of mononuclear phagocytes to deliver imaging agents to regions of hypoxia/ischaemia to increase the resolution of detection of said imaging agent either through localisation or by use of hypoxia enhanced imaging agent.